Elisa Chisci

Simultaneous overexpression of human E5NT and ENTPD1 protects porcine endothelial cells against H2O2-induced oxidative stress and cytotoxicity in vitro

Abstract Ischemia‐reperfusion injury (IRI) and oxidative stress still limit the survival of cells and organs in xenotransplantation models. Ectonucleotidases play an important role in inflammation and IRI in transplantation settings. We tested the potential protective effects derived by the co‐expression of the two main vascular ectonucleotidases, ecto‐5’‐nucleotidase (E5NT) and ecto nucleoside triphosphate diphosphohydrolase 1 (ENTPD1), in an in vitro model of H2O2‐induced oxidative stress and cytotoxicity. We produced a dicistronic plasmid (named pCX‐DI‐2A) for the co‐expression of human E5NT and ENTPD1 by using the F2A technology. pCX‐DI‐2A‐transfected porcine endothelial cells simultaneously overexpressed hE5NT and hENTPD1, which were correctly processed and localized on the plasma membrane. Furthermore, such co‐expression system led to the synergistic enzymatic activity of hE5NT and hENTPD1 as shown by the efficient catabolism of pro‐inflammatory and pro‐thrombotic extracellular adenine nucleotides along with the enhanced production of the anti‐inflammatory molecule adenosine. Interestingly, we found that the hE5NT/hENTPD1 co‐expression system conferred protection to cells against H2O2‐induced oxidative stress and cytotoxicity. pCX‐DI‐2A‐transfected cells showed reduced activation of caspase 3/7 and cytotoxicity than mock‐, hE5NT‐ and hENTPD1‐transfected cells. Furthermore, pCX‐DI‐2A‐transfected cells showed decreased H2O2‐induced production of ROS as compared to the other control cell lines. The cytoprotective phenotype observed in pCX‐DI‐2A‐transfected cells was associated with higher detoxifying activity of catalase as well as increased activation of the survival signaling molecules Akt, extracellular signal‐regulated kinases 1/2 (ERK1/2) and p38 mitogen‐activated protein kinase (MAPK). Our data add new insights to the protective effects of the combination of hE5NT and hENTPD1 against oxidative stress and constitute a proof of concept for testing this new genetic combination in pig‐to‐non‐human primates xenotransplantation models. Graphical abstract Figure. No Caption available. Highlightshuman E5NT and ENTPD1 genes were correctly co‐expressed in porcine endothelial cells.hE5NT/hENTPD1 co‐expression led to enhanced production of adenosine.hE5NT/hENTPD1 co‐expression protected cells against H2O2‐induced cytotoxicity.hE5NT/hENTPD1 co‐expression lead to less ROS formation and more catalase activity.hE5NT/hENTPD1‐transfected cells showed more activation of Akt, ERK1/2 and p38 kinases.

Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro

Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.

CD73 Regulates Stemness and Epithelial-Mesenchymal Transition in Ovarian Cancer-Initiating Cells

Summary Cancer-initiating cells (CICs) have been implicated in tumor development and aggressiveness. In ovarian carcinoma (OC), CICs drive tumor formation, dissemination, and recurrence, as well as drug resistance, thus accounting for the high death-to-incidence ratio of this neoplasm. However, the molecular mechanisms that underlie such a pathogenic role of ovarian CICs (OCICs) remain elusive. Here, we have capitalized on primary cells either from OC or from its tissues of origin to obtain the transcriptomic profile associated with OCICs. Among the genes differentially expressed in OCICs, we focused on CD73, which encodes the membrane-associated 5′-ectonucleotidase. The genetic inactivation of CD73 in OC cells revealed that this molecule is causally involved in sphere formation and tumor initiation, thus emerging as a driver of OCIC function. Furthermore, functional inhibition of CD73 via either a chemical compound or a neutralizing antibody reduced sphere formation and tumorigenesis, highlighting the druggability of CD73 in the context of OCIC-directed therapies. The biological function of CD73 in OCICs required its enzymatic activity and involved adenosine signaling. Mechanistically, CD73 promotes the expression of stemness and epithelial-mesenchymal transition-associated genes, implying a regulation of OCIC function at the transcriptional level. CD73, therefore, is involved in OCIC biology and may represent a therapeutic target for innovative treatments aimed at OC eradication.

A Novel KCNJ2 Mutation Identified in an Autistic Proband Affects the Single Channel Properties of Kir2.1

Inwardly rectifying potassium channels (Kir) have been historically associated to several cardiovascular disorders. In particular, loss-of-function mutations in the Kir2.1 channel have been reported in cases affected by Andersen-Tawil syndrome while gain-of-function mutations in the same channel cause the short QT3 syndrome. Recently, a missense mutation in Kir2.1, as well as mutations in the Kir4.1, were reported to be involved in autism spectrum disorders (ASDs) suggesting a role of potassium channels in these diseases and introducing the idea of the existence of K+ channel ASDs. Here, we report the identification in an Italian affected family of a novel missense mutation (p.Phe58Ser) in the KCNJ2 gene detected in heterozygosity in a proband affected by autism and borderline for short QT syndrome type 3. The mutation is located in the N-terminal region of the gene coding for the Kir2.1 channel and in particular in a very conserved domain. In vitro assays demonstrated that this mutation results in an increase of the channel conductance and in its open probability. This gain-of-function of the protein is consistent with the autistic phenotype, which is normally associated to an altered neuronal excitability.

The Role of Hydrogen Peroxide in Redox-Dependent Signaling: Homeostatic and Pathological Responses in Mammalian Cells

Hydrogen peroxide (H2O2) is an important metabolite involved in most of the redox metabolism reactions and processes of the cells. H2O2 is recognized as one of the main molecules in the sensing, modulation and signaling of redox metabolism, and it is acting as a second messenger together with hydrogen sulfide (H2S) and nitric oxide (NO). These second messengers activate in turn a cascade of downstream proteins via specific oxidations leading to a metabolic response of the cell. This metabolic response can determine proliferation, survival or death of the cell depending on which downstream pathways (homeostatic, pathological, or protective) have been activated. The cells have several sources of H2O2 and cellular systems strictly control its concentration in different subcellular compartments. This review summarizes research on the role played by H2O2 in signaling pathways of eukaryotic cells and how this signaling leads to homeostatic or pathological responses.

Abstract LB-282: Two different strategies of delivery CRISPR/Cas9 system to gene edit rs4644 SNP inLGALS3gene

Galectin-3 is a glycoprotein of 31KDa with a chimeric structure. It is encoded by a single gene, LGALS3, located on chromosome 14. Previous studies showed a relation between the single nucleotide polymorphism (SNP) (rs4644, c.191C>A, p.Pro64His) and the risk of cancer. In literature, some data show different results indicating that SNP could have different roles in base on the tissues. The aim of our study is to create isogenic cell lines that differ only for SNP permitting further studies about the function of SNP in different cancers. The “generation” of the cell lines is performed with a Clustered Regularly Interspaced Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9) system. We have used two different strategies to deliver the system. In the first strategy, our aim was the reduction of the problem of efficiency of transfection that could depend on different cell lines. In fact, we have used the lentivirus method to deliver all components of the CRISPR/Cas9 on Nthy-Ori (normal thyroid tissue) and HCT 116 +/+ (colorectal cancer tissue) cells. Two different vectors (pCW-Cas9 and pLXgRNA) are used to build up the inducible CRISPR/Cas9 system and HR410PA-1 vector (transfected by Lipofectamine 3000) is used to knock-in. HR410PA-1 is a particular vector that facilities the homologous recombination (HR) to repair the double strand break. Moreover, to improve HR, we use also Scr7, which is an inhibitor of DNA IV ligase. In the second approach, we have used the double nickase system using two modified Cas9 vectors (SpCas9D10a) that produce only a single strand break. In this way, the problem of off-targets is notably reduced. In this approach, the knock-in is due to or single-stranded oligodeoxynucleotides (ssODNs) or HR410PA-1 vector. The first results show that some of the transfected cells are edited in a correct way. In the further steps, we will isolate the “positive clones” using the selection cassette in HR410PA-1 vector (GFP protein and/or puromycin) or the single cell dilution assay (1 cell/well) for the transfected cells with ssODNs. Citation Format: Alda Corrado, Irene Lepori, Simona Miglietta, Simone Batoni, Marianna Vitiello, Monica Evangelista, Elisa Chisci, Roberto Giovannoni, Laura Poliseno, Federica Gemignani, Stefano Landi. Two different strategies of delivery CRISPR/Cas9 system to gene edit rs4644 SNP in LGALS3 gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-282. doi:10.1158/1538-7445.AM2017-LB-282